Chimeric interleukin 5-receptor/immunoglobulin polypeptides

ABSTRACT

Chimeric polypeptides encoded by a DNA sequence having a first DNA subsequence coding for a fragment of at least one of the  alpha - and/or  beta -chain of the human interleukin-5 receptor, and a second DNA subsequence coding for the constant domains of a heavy- or a light-chain of a human immunoglobulin, or a fragment thereof are useful in treating illnesses with demonstrated eosinophilia.

This is a division of application Ser. No. 07/947,130, filed Sep. 16,1992 now U.S. Pat. No. 5,455,337.

FIELD OF THE INVENTION

The invention is directed to polypeptides and a DNA sequence coding fora fragment of one or both of the α- and/or β-chain of the humaninterleukin-5 receptor and coding for the constant domains of a heavy-or a light-chain of a human immunoglobulin, or a fragment thereof.

BACKGROUND OF THE INVENTION

Interleukin-5 (IL-5 or IL5) is a lymphokine secreted by T cells and mastcells and biologically activates B cells and eosinophils. The activityon B cells seems to be restricted to the murine system. No detectableactivity can be found in a panel of human B-cell activation ordifferentiation assays. [Clutterbuck et al., Eur. J. Immunol. 17,1743-1750 (1987)].

In murine hematopoiesis, IL-5 is a selective signal for theproliferation and differentiation of the eosinophilic lineage [Yamaguchiet al., J. Exp. Med. 167, 43-56 (1988)]. In this respect, IL-5 functionshows analogies with colony-stimulating factors for other myeloidlineages. Also, human (h) IL-5 is very potent in the activation of humaneosinophils [Lopez et al., J. Exp. Med. 167, 219-224 (1988); Saito etal., Proc. Natl. Acad. Sci USA 85, 2288-2292)]. A good discussion of theroles of IL-5 and eosinophils in disease is provided by Sanderson, C.J., Blood, Vol. 79, No. 12 (June 15), 1992, pp. 3101-3109.

Interleukin 5 mediates its activity through a cell membranereceptor-complex. This complex has been characterized physicochemicallyin both the murine and human system. Mouse pre B cell lines depending onIL5 for their growth have been developed from bone marrow and are usedfor IL5-receptor analysis [Rolink et al., J. Exp. Med. 169, 1693-1701(1989)]. The human IL5-receptor (hIL-5R) can be studied on a subclone ofthe promyelocytic cell line HL60 induced towards eosinephildifferentiation [Plaetinck et al., J. Exp. Med. 172, 683-691 (1990)].

Eosinophilic differentiation is initiated using sodium butyrate. Onlyhigh affinity (Kd=30 pM) IL5 binding sites can be found on these cells.However cross-linking studies reveal the presence of two polypeptidechains of the receptor involved in IL5 binding, with molecular massesclosely resembling the murine IL5R-α- and -β chains.

Increased half-life in vive has been shown for example, for chimericpolypeptides consisting of the first two domains-or parts thereof of thehuman CD4-molecule and different domains of the constant regions of theheavy chain or the light chain of a mammalian immunoglobulin (seeTraunecker et al., Nature 331, 84-86 [1988] and European PatentApplication 90107393.2, Publication No. 394,827).

The specification of European Patent Application 90107393.2, therelevant portions of which are described later in the specification,contains data with respect to the use of pSV-2-derived vectors for theexpression of chimeric proteins as well as the construction of vectorsfor the expression of such chimaeric proteins with other immunoglobulinfragments.

As described above, sources for DNA sequences coding for constantdomains of human immunoglobulins are known in the state of the art anddisclosed, for example, in EP 394,827 or are described for example byEllison et al., Nucl. Acid Res. 10, 4071-4079 (1982) for IgG1, or Hucket al., Nucl. Acid Res. 14, 1779-1789 (1986) for IgG3.

SUMMARY OF THE INVENTION

The invention is directed to a DNA sequence which comprises acombination of two DNA subsequences, with one of the subsequences cedingfor a fragment of the α- and/or the β-chain of the hIL5R. The hIL5Rfragment, or combination of fragments, is capable of binding hlL-5, andthe other subsequence codes for part or all constant domains of thehuman immunoglobulin heavy- or light-chains.

The invention additionally pertains to vectors comprising such DNAsequences, especially such vectors capable of expression in eukaryotichost cells. The invention also relates to prokaryotic or eukaryotic hostcells transformed with such vectors.

The present invention is also concerned with the recombinant chimericpolypeptides, ceded by such DNA sequences, as well as their use,especially for the treatment of illnesses having demonstratedeosinophilia, for example chronic asthma, and helmith infections. Asoluble human IL5Rα-chain (shlL5Rα) would be advantageous as an IL-5antagonist in chronic asthma or other disease states with demonstratedeosinophilia. In addition the shIL5Rα or the α-chain itself or the wholehigh affinity receptor, consisting of the α-chain and the β-chain[Tavernier et al., Cell 66. in press (1991)] could be used as a tool forscreening for IL-5 antagonists. Of course, the invention also includessuch proteins in which the amino acid sequences are deletedor-exchanged, so that the activity of the proteins is not significantlyaltered. Amino acid exchanges in proteins and peptides which do notgenerally alter the activity of such molecules are known in the state ofthe art and are described, for example, by H. Neurath and R. L. Hill in"The Proteins" (Academic Press, New York, 1979, see especially FIG. 6,page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Ash, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well asthese in reverse.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the plasmids pHT4-YK12, pCD4-Mμ and pCD4-Mγ2a. Boxes referto pieces of cDNA equivalent to specific parts of exons [see Example 14]or exons themselves used for the construction of the plasmids whereinthe white boxes refer to DNA coding for the leader region (L) and allfour (1,2,3,4) extracellular domains of the human CD4-molecule. Theblack box refers to the exons coding for the immunoglobulin κlight-chain constant region (cκ). The dotted boxes refer to the exonscoding for the hinge (H) and the second and third domains of the mouseIgG_(2a) constant regions (C2, C3) respectively. The striped boxes referto the exons coding for the second, third and fourth domains for themouse IgM constant regions respectively. Further abbreviations refer to:B=BamHI restriction site, R=EcoRI restriction site, S=Sa1I restrictionsite, Ss=SstI restriction site, St=Stul restriction site, X=XbaIrestriction site, Xh=XhoI restriction site, amp=ampicillin resistancegene, gpt=E. coli xanthine-guanine phosphoribosyltransferase gene, κprom=Igκ promotor, κENH=Igκ enhancer, H.CENH=Ig heavy chain enhancer.

FIG. 2 shows plasmids pHT4-YK12 (see FIG. 1), pCD4-Hμ and pCD4-Hγ1. Inthese latter plasmids, the boxes represent the human IgG and IgM regionscorresponding to the mouse exons of FIG. 1. "P" refers to a PstIrestriction site and "Ha" to a HaeII restriction site, "PV" to a PvuIIrestriction site and "Sm" to a SmaI restriction site. All other symbolsand abbreviations have the same meaning as in FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed, in part, to a DNA sequence which comprises acombination of two DNA subsequences, with one of the subsequences codingfor a fragment-of the α- and/or the β-chain of the hlL5R. The hlL5Rfragment, or combination of fragments, is capable of binding hIL5, andwhereby a fragment of the soluble hIL5Rα-chain (shlL5Rα) and especiallysuch a fragment with the whole or a part of the sequence as shown in SEQID NO: 1 is preferred. The other subsequence codes for constant domainsof the human immunoglobulin heavy- or light-chains. The heavy chains,especially all domains except the first domain of the constant domain ofhuman immunoglobulins such as IgG, IgA, IgM or lgE and specifically IgG,for example IgG1 and IgG3 are preferred.

It is furthermore understood that a DNA sequence coding for a fragmentof the α-chain of the hIL5R which fragment binds hIL5 comprises also DNAsequences which hybridize under stringent. hybridization conditions to aDNA sequence as shown in SEQ ID NO: 1 which DNA sequence codes for aprotein which is capable of binding hIL5. A man skilled in the art willeasily be able to define such stringent hybridization conditions basedon the DNA-sequence shown in FIG. 1 and according to standard knowledgein the state of the art and disclosed for example in Sambrook et al."Molecular Cloning", 2nd. ed., Cold Spring Harbor Laboratory Press(1989). The DNA sequences of the invention also comprise DNA sequenceswhich hybridize to the DNA sequence which is complementary to the DNAsequence shown in SEQ ID NO: 1. In addition, the DNA sequence whichcodes for a fragment of the α-chain of the hIL5R fragment which fragmentis capable of binding hIL5 also comprises DNA sequences which, becauseof the degeneracy of the genetic code, do not hybridize with thesequence of SEQ ID NO: 1, or its complement, but which code forpolypeptides having exactly the same amino acid sequence as the fragmentof the α-chain of the human IL5R.

It is furthermore understood that for the purpose of the presentinvention-the chimeric polypeptides can be in a dimeric form, namelyconsisting either of two subunits whereby each subunit comprises afragment of the α-chain of the IL5R which binds hIL5 or of two subunitswhereby one of the two subunits comprises a fragment of the α-chain ofthe IL5R and the other subunit comprises a fragment of the β chain ofthe IL5R so that the dimeric polypeptide binds hIL5.

Cloned DNA can be obtained in the form of genomic DNA isolated from agenomic library by methods well known in the art and described forexample by Maniatis, T., et al. in "Molecular Cloning." For example,specific obligonucleotide probes can be prepared on the at least partialknowledge of the amino acid sequence encoded by the specific genomic DNA(exons) to be isolated or the least partial knowledge of its nucleicacid sequence. Such sequence information can be obtained in principlefrom any sequence data base, for example, Genbank (Intelligenetics,California, USA) and Vecbase (University of Wisconsin, BiotechnologyCentre, Madison, Wis., USA), or more specifically, for example, for thehuman Ig μ from Rabbits, T. H., et al., Nucleic Acid Res., 9:4509-4524(1981); for the human IgY1 from Ellison, J. W., et al., Nucleic AcidRes., 10:4071-4079 (1982); for the human Ig γ3 heavy chain gene fromHuch, S., et al., Nucleic Acid Res., 14:1779-1789 (1986); and for thehuman Igα1 and Igα2 heavy chain gene from Flanagan, J. G., et al., Cell,36:681-688 (1984).

The cloning of a DNA sequence coding for the α-chain of the hIL5R can beachieved in the following manner. Murine cell lines which contain themurine IL-5-receptor (mlL5R) in membrane-bound form, can be cultivatedaccording to methods known in the art or as specifically described, forexample, in Example 2. Such cells can then be harvested bycentrifugation, lysed and a membrane extract can be prepared by using asuitable detergent, for example Triton-X-100. For the isolation of theα-chain of mlL5R, the membrane extract, cleared by centrifugation, canbe passed over an immuneaffinity matrix. The corresponding antibodiesfor such an immunematrix namely the ones to the α-chain of the mIL5R canbe prepared and coupled to an appropriate matrix by methods well knownin the art or as specifically described, for example, in Examples 1-3.The α-chain of the mIL5R can be further purified by sodiumdodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and blottedto an appropriate matrix.

The thus-purified murine IL-5-receptor chain can be characterized bymethods of peptide chemistry which are known in the state of the art,such as, for example, N-terminal amino acid sequencing or enzymatic aswell as chemical peptide cleavage. Fragments obtained by enzymatic orchemical cleavage can be separated according to usual methods such as,for example, HPLC and can themselves be subjected to further N-terminalsequencing.

Starting from the so-obtained amino acid sequence information,oligonucleotides can be produced according to methods known in the stateof the art [see, for example, Sambrook et al., supra] taking intoconsideration the degeneration of the genetic code.

cDNA or genomic DNA libraries can be produced according to methods knownin the art [Sambrook et al., supra.], whereby cDNA libraries on thebasis of an mRNA-preparation from cell lines expressing with or withoutinduction murine or human IL5R, for example as specifically described inExample 4, are preferred. Such libraries can then be screened byoligonucleotides [Sambrook et al., supra]. Once a specific clone hasbeen identified in such a manner, the phage harboring the desired DNAsequence of the invention can be isolated [Sambrook et al., supra] andthe corresponding inserts characterized by restriction enzyme cleavagepattern analysis or sequencing according to standard procedures[Sambrook et al., supra]. It is understood that DNA sequenceshybridizing under stringent hybridization conditions to those of thepresent invention and coding for proteins which bind IL5 can be employedfor the purpose of the present invention. Such DNA sequences can beprepared for example by mutagenesis methods known in the art [see, forexample, Sambrook et al., supra] starting from the correspondingnon-mutated DNA sequences. Furthermore, the well-known polymerase chainreaction (PCR) can be used for the preparation of DNA sequences of thepresent invention as described in detail in examples 12 and 13.Stringent hybriditation conditions can be determined by a man skilled inthe art by standard procedures as given, e.g. by [Sambrook et al.,supra].

On the basis of the thus-determined DNA sequences and of the alreadyknown DNA sequences for certain receptors, those partial DNA sequenceswhich code for a soluble receptor subunit can be determined and cut outfrom the complete DNA sequence using known methods, see Sambrook et al.,supra and Maliszewski and Fanslow, Tibtech., 8, 324-329 (1990).

The complete DNA sequence or such partial DNA sequences can then beintegrated using known methods into expression vectors described in thestate of the art for their amplification and/or expression inprokaryotes [Sambrook et al., supra]. Suitable prokaryotic hostorganisms are, for example, gram-negative and gram-positive bacteriasuch as, for example, B. subtilis strains or E. coli strains such as E.coli HB 101 [ATCC No. 33 694] or E. coli W3110 [ATCC No. 27 325] and E.coli MC1061 [Casadabam and Cohen, J. Mol. Biol. 138, 179-207 (1980)].The latter two harboring plasmid "p3" [Sambrook et al., supra] in casethe pCDM8-type vectors like πV X or pshlL5Rα (see Example 9) will beamplified.

Furthermore such DNA sequences can be integrated using known methodsinto suitable vectors for expression in eukaryotic host cells, such as,for example, yeast, insect cells and mammalian cells.

A typical expression vector for mammalian cells contains an efficientpromoter element in order to produce a good transcription rate of theDNA. Also included in the vector is the DNA sequence to be expressed,and DNA coding signals for an efficient termination and polyadenylationof the transcript. Additional elements which can be used are "enhancers"which lead to again intensified transcription, and sequences which forexample can bring about a longer half life of the mRNA. For theexpression of nucleic acid sequences in which the endogenous sequencefragment coding for a signal peptide is missing, there can be usedvectors which contain such suitable sequences which code for signalpeptides of other known proteins. See, for example, the vector pLJ268described by Cullen, B. R. in Cell 46, 973-982 (1986) as well as Sharma,S. et al. in "Current Communications in Molecular Biology", edt. byGething, M. J., Cold Spring Harbor Lab. (1985), pages 73-78.

Most of these vectors which are used for a transient expression of aparticular DNA sequence in mammalian cells contain the replicationorigin of the SV40 virus. In cells which express the T-antigen of thevirus (for example COS cells), these vectors are reproduced abundantly.A transient expression as described for example in Example 10 is,however; not limited to COS cells. In principle, any transfectablemammalian cell line can be used for this purpose. Signals whichcan-bring about a strong transcription are for example m the early; andlate promoters of SV40, the promoter and enhancer of the "majorimmediate-early" gene of HCMV (human cytomegalovirus), the LTR's ("longterminal repeats") of retroviruses such as, for example, RSV, HIV andMMTV. There can, however, also be used signals of cellular genes such asfor example the promoters of the actin and collagenase genes.

Alternatively, however, stable cell lines which have the specific DNAsequence integrated into the genome (chromosome) also are suitable. Forthis, the DNA sequence is cotransfected together with a selectablemarker, for example neomycin, hygromycin, dihydrofolate reductase (dhfr)or hypoxanthin guanine phosphoribosyl transferase (hgpt) using methodswhich are per se known in the art. The DNA sequence stably incorporatedin the chromosome can also be amplified abundantly. A suitable selectionmarker for this is, for example, dihydrofolate reductase (dhfr).Mammalian cells, for example, chinese hamster ovary (CHO) cells, whichcontain no intact dhfr gene, are thereby incubated with increasingamounts of methotrexate after transfection has been effected. In thismanner cell lines which contain more than a thousand copies of thedesired DNA sequence can be obtained.

Mammalian cells which can be used for expression are for example cellsof the human cell lines Hela [ATCC CCL2] and 293 [ATCC CRL 1573] as wellas 3T3 [ATCC CCL 163] and L cells, for example [ATCC CCL 149], (CHO)cells [ATCC CCL 61], BHK [ATCC CCL 10] cells as well as the CV 1 [ATCCCCL 70] and the COS cell lines [ATCC CRL 1650, CRL 1651].

Suitable expression vectors include, for example, vectors such aspBC12MI [ATCC 67 109], pSV2dhfr [ATCC 37 146], pSVL [Pharmacia, Uppsala,Sweden], pRSVcat [ATCC 37 152], pMSG [Pharmacia, Uppsala, Sweden] andpCDM8 type plasmids like for example pshIL5Rα [see Example 7] which hasbeen deposited transformed in E. coli MC1061 (harboring plasmid p3)under the conditions of the Budapest Treaty for patent purposes at theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) inBraunschweig, Federal Republic of Germany on Apr. 17, 1991 underaccession number DSM 6479. The plasmid pshIL5Rα can be isolated from thedeposited transformed E. coli as known in the art and described forexample in detail in Example 9.

For the expression of the chimeric polypeptides of the presentinvention, there can be used pSV2-derived vectors [see for exampleGerman, C. in "DNA Cloning" Vol. II., edt. by Glover, D. M., IRL Press,Oxford, 1985] like pCD4-Hμ (DSM 5315), pCD4-Hγ1 (DSM 5314) and pCD4-Hγ3(DSM 5523) which have been deposited at the Deutschen Sammlung vonMikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG, andwhich are described in detail in European Patent Application No.90107393.2, Publication No. 394,827. For the purpose of the presentinvention, the CD4 coding part in the vectors of the EPA applicationmust be replaced by a DNA sequence coding for a fragment of the α-and/or β-chain of the hIL5R which binds hIL5. The replacement of the CD4region with the hIL5R fragment is performed by methods known per se inthe art and described for example in Sambrook et al., supra. Ifdesirable, the specific immunoglobulin coding region in the vectorsobtained also can be replaced by a DNA sequence coding for the desiredimmunoglobulin. Preferred vectors for the expression of the chimericpolypeptides of the present invention are pCDM8 type vectors like forexample pshlL5Rα for the expression of fragments of the α-chain of theIL5R containing chimeric polypeptides (see examples 12 and 13).

The manner in which these cells are transfected depends on the chosenexpression system and vector system. An overview of these methods is tobe found, for example, in Pollard et al., "DNA Transformation ofMammalian Cells" in "Methods in Molecular Biology", Nucleic Acids Vol.2, 1984, Walker, J. M., ed, Humana, Clifton, N.J. Further methods are tobe found in Chen and Okayama "High-Efficiency Transformation ofMammalian Cells by Plasmid DNA", Molecular and Cell Biology 7,2745-2752, 1987 and in Felgner Felgner et al., "Lipofectin: A highlyefficient, lipid-mediated DNA-transfection procedure", Proc. Nat. Acad.Sci. USA 84, 7413-7417, 1989.

The baculovirus expression system, which has already been usedsuccessfully for the expression of a series of proteins (for an overviewsee Luckow and Summers, Bio/Technology 6, 47-55, 1988), can be used forthe expression in insect cells. Recombinant proteins can be produced inauthentic form or as fusion proteins. The thus-produced proteins canalso be modified such as, for example, glycosylated (Smith et al., Proc.Nat. Acad. Sci. USA 82, 8404-8408, 1987). For the production of arecombinant baculovirus which expresses the desired protein there isused a so-called "transfer vector". Under this there is to be understooda plasmid which contains the heterologous DNA sequence under the controlof a strong promoter, for example that of the polyhedron gene, wherebythis is surrounded on both sides by viral sequences. The transfer vectoris then transfected into the insect cells together with DNA of the wildtype baculovirus. The recombinant viruses which result in the cells byhomologous recombination can then be identified and isolated accordingto known methods. An overview of the baculovirus expression system andthe methods used therein is to be found in Luckow and Summers, "A Manualof Methods for Baculovirus Vectors and Insect Cell Culture Procedures",Texas Agricultural Experimental Station, Texas A & M University,Bulletin No. 1555, 2nd edition, 1988. It is understood that for thepractice of the present invention when using the baculovirus expressionsystem, DNA-sequences coding for the immunoglobulin part have to be inthe form of a cDNA.

The chimeric polypeptides of the invention can then be purified from thecell mass or the culture supernatants according to methods of proteinchemistry which are known in the state of the art, such as, for example,precipitation for example with ammonium sulfate, dialysis,ultrafiltration, gelfiltration, ion-exchange chromatography, SDS-PAGE,isoelectrie focusing, affinity chromatography like immunoaffinitychromatography, HPLC in-normal or reverse phase systems or the like.

The chimeric polypeptides of the invention as well as theirphysiologically compatible salts, which can-be manufactured according tomethods which are known in the state of the art, can also be used forthe treatment of illnesses in which IL-5 is involved in their courseand/or the production of corresponding pharmaceutical preparations. Forthis purpose, one or more of the compounds, where desired or required incombination with other pharmaceutically active substances, can beprocessed in a known manner with the usually used solid or liquidcarrier materials. The dosage of such preparations can be effectedhaving regard to the usual criteria in analogy to already usedpreparations of similar activity and structure. Such pharmaceuticalpreparations and the use of the compounds of the present invention fortherapeutical purposes are also an object of the present invention.

The following Examples are intended to illustrate details of theinvention, without thereby limiting it in any manner. Unless indicatedotherwise, the Examples were carried out as written. Unless indicatedotherwise, all methods used below are standard methodology according toSambrook et al. (1989), Molecular Cloning. A Laboratory Manual (2ndedn). Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.

EXAMPLE 1 Production of monoclonal antibodies against the murine IL5R

Immunization was carried out generally as described by A. Rolink et al.,J. Exp. Med., 169, 1693-1701 (1989). That is, at day 0, 2×10⁷ B13 cells(Rolink et al., supra) were washed with phosphate buffered saline(PBS-A), mixed with complete Freund's adjuvant (CFA) and injected intothe hind footpath of Wistar rats. This was repeated without Freund'sadjuvant (FA) on day 5 and 7. On day 8, regional lymph nodes wereremoved and a cell suspension was prepared. These cells were fused usingPEG 1500 (Boehringer) with Sp2/0-Ag14 cells [ATCC CRL 1581] at a ratioof 5:1.5. Cells were plated in microtiterplates in the presence of 500pg/ml of recombinant hIL-6 [Haegemann et al., Europ. J. Biochem. 159,625-632 (1986)]. The next day, the same volume of medium containing a 2×conc. of aminopterin was added for selection of hybrid cells. Cells wererefed at day 8 with medium without aminopterin. Hybridomas were selectedon the ability of their supernatant to inhibit the mIL5 [Tavernier, J.et al., DNA 8, 491-501 (1989)] or a mouse interleukin-3 (mIL3) drivenproliferation of B13 cells (measured by a ³ Hdeoxy-cytidin incorporationassay as known in the art). Conditioned medium from WEHI-3 cells (ATCCNo. TIB68) was used as a source of mIL3. Supernatants demonstratinginhibiting activity were retested in a competition-binding assay withradiolabeled (according to methods known in the art) mIL5 or "R52" (amonoclonal antibody recognizing only the β-chain of the IL-5-R, seeRolink et al., supra) on B13 cells. Monoclonal antibodies directed onlyto the α-chain of the mIL-5-R were identified on their ability to almostcompletely inhibit mIL5 binding and by immunprecipitation of thecorresponding mIL-5-R chain. Selected hybridomas were recloned by thewell-known limiting dilution method.

EXAMPLE 2 Immunoaffinity purification of the mIL5R-β-chain

B13 cells were grown in large spinner flasks in Iscove's modifiedDulbecco's medium (Gibco Laboratories, Grand Island N.Y., USA)containing 5% fetal calf serum, 2 mM L-glutamine, 50 μg/ml gentamycin,and 100 units/ml recombinant mouse IL-5, to a density of 2×10⁶ cells/mi.Cells from 10 l cultures were concentrated by centrifugation, washedwith PBS and lysed in 200 ml PBS containing 1% Triton-X-100 and acocktail of protease inhibitors (1 mM PMSF, 10 mM benzamidine. HCl, 100U/ml aprotinin). After 10 min on ice, the lysate was centrifuged for 10min at 1000× g and cleared by ultracentrifugation (100.000× g) for 90min at 4° C. The supernatant was diluted with NaCl to a finalconcentration of 0.5M, and used for purification. "R52" was covalentlybound to protein G-Sepharose 4 Fast Flow (Pharmacia, LKB BiotechnologyAB, Uppsala, Sweden) according to Schneider et al. [J. Biol. Chem. 257,10766 (1982)], at a concentration of 5 mg/ml gel. Two hundred ml lysateof B13 cells was passed at 4° C. over 2 ml protein G-Sepharose 4 FastFlow followed by 2 ml R52-linked protein G-Sepharose 4 Fast Flow bothpacked in a 1 cm diameter column. The flow through was then reloaded onboth columns. The gel was washed extensively (100 ml) with a buffercontaining 50 mM Tris-HCl (pH8.2), 1 mM EDTA, 0.5M NaCl, 0.5% NP40,followed by 10 ml 0.1% (NP40). Next, the retained proteins were elutedin 4 ml 50 mM diethylamine (pH11) containing 0.1% Nonidet P40 (NP40),neutralized by addition of 1M NaH₂ PO₄ and concentrated bylyophilization. The purity was assessed by SDS-PAGE and Coomassiestaining of 2.5% of the eluate.

EXAMPLE 3 Immunoaffinity of the murine IL5R α-chain

B13 cell lysates from 2×10¹⁰ cells (run through fractions of the"R52"-immunoaffinity column used to purify the β-chain doublet accordingto Example 2) were mixed overnight by 4° C. with 2 ml hydrazide avidgelAX (Bioprobe Int. Inc.) armed with 10 mg mAbs recognizing the mIL5Rα-chain. The gel was then poored into a column, and after extensivewashing (50 mM Tris. HCl, pH 8.2, 1 mM EDTA, 0.5M NaCl, 0.5% NP40;followed by 0.1% NP40 in H₂ O) elution was performed using 50 mMdiethylamine, pH11, 0.01% NP40. Selected fractions were immediatelylyophilized and resuspended in 2× Laemmli buffer, in the presence ofβ-mercaptoethanol. Samples were run through a 1.5 mm 10% PAGE-SDS gel.The gel was fixed in 10% HAc, 30% methanol and stained with CoomassieBrilliant Blue. Slices containing the 60 kDa mIL5R α-chain were treatedwith SDS-buffer, sliced further and electrophoresed in a new PAGE-SDSgel.

After transfer to an Immobilon-P membrane (Millipore Corp.), andstaining with amido black, the 60 kDa band was excised and in situdigested with trypsin. Peptides were separated on a C4-reversed-phasecolumn and subjected to sequence analysis using a 470A-type gas-phasesequenator equipped with an on-line 120A-type PTH-amino acid analyser(Applied Biosystems Inc., Foster City, Calif.). Amino acid sequences(standard abbreviations of amino acids) and the sequences ofcorresponding sets of oligonucleotide probes, synthesized according tomethods known in the art, are shown below: ##STR1##

EXAMPLE 4 Construction of unidirectional λGT11 cDNA libraries

1. Murine pre-B cell B13 cDNA library

mRNA was extracted from B13 cells using the "fast-track" mRNA isolationsystem (Invitrogen Corp.). Using this protocol, poly(A)⁺ mRNA wasdirectly isolated from cell lysates using oligo(dT) cellulose; yieldswere around 50 μg per 10⁸ cells. 5 mg poly(A)⁺ mRNA was reversetranscribed using an oligo dT-Not1 primer-adaptor (5'-AATTCGCGGCCGC(T)₁₅-3' [SEQ ID NO: 16], Promega Corp.) and cloned Moloney Murine LeukemiaVirus RNaseH⁻ Reverse Transcriptase (BRL Life Technologies, Inc.). EcoRltinkered double stranded cDNA was made using described procedures[Sambrook et al., supra]. Not1 cleavage was used to generate a unique 3'sticky-end, and cDNAs were size selected (>1.000 bp) on a 1% agarosegel. After elution using the "gene clean" protocol (BIO 101 Inc.), cDNAswere ligated into the EcoRl-Not1 arms of the λgt11 Sfi-Not vector(Promega Corp.). After in vitro packaging, around 40×10⁶ recombinantphages were obtained.

2. Human, HL60 clone (butyrate induced) cDNA library

Prior to mRNA purification, butyrate induced HL60 clone 15 cells[Fischkoff, Leukemia Res. 12, 679-686 (1988); Plaetinck et al., J. Exp.Meal. 172, 683-691 (1990); HL60: ATCC-No. CCL 240] were checked forproper ¹²⁵ I-hIL5 binding (around 2000 binding sites per cell). The sameprotocols as for the murine pre-B cell B13 cDNA library, above, wereused, and a comparable yield of recombinant phage was obtained.

EXAMPLE 5 Screening of murine and human cDNA libraries

Two sets of oligonucleotide probes "Oligonucleotide 1" and"Oligonucleotide 2", see Example 3, were used for screening underdifferent hybridization conditions, dependent on the type of probe used,by methods known in the art [Sambrook et al., supra]. Results arepresented in the scheme below:

1. Two cDNA clones (λgt11-mIL5Rα2,3) were selected from part of themurine cDNA library (1.2×10⁶ plaques were screened), on the basis ofhybridization with both sets of oligonucleotide probes. For thatpurpose, plaque lifts were prepared as described using Biodyne Atransfer membranes (Pall), [Sambrook et al., supra]. Oligonucleotide 1was radioactively labeled by kinasing [Sambrook et al., supra] and washybridized under "intermediate stringency" hybridization conditions, asdescribed below. Oligonucleotide 2 was radioactively labeled by kinasing[Sambrook et al., supra] and was hybridized under "low stringency"hybridization conditions as described below.

2. One cDNA clone (λgt11-hIL5Rα8) was selected from part of the humancDNA library (2.4×10⁶ plaques were screened), on basis of hybridizationwith both "oligonucleotide 1" and the cDNA insert, which was derived bymethods known in the art, from the murine λgt 11mIL5Rα2.

Oligonucleotide 1 was radioactively labeled by kinasing [Sambrook etal., supra] and was hybridized under "low stringency" hybridizationconditions. The cDNA insert form λgt11mIL5Rα2 was radioactively labeledby random labeling [Sambrook et al., supra] and was hybridzed under"intermediate stringency" hybridization conditions.

3. Five additional cDNA clones (λgt11-hIL5Rα11→15) were selected fromhalf of the human cDNA library screened as in 2. above, using themIL5Rα2 cDNA probe. Hybridization was under "intermediate stringency"conditions.

4. Thirty-six additional cDNA clones (λgt11-hIL5Rα16→51) were selectedfrom the other half of the human cDNA library screened as in 2. aboveusing the hIL5Rα8-cDNA probe. Hybridization was under "high stringency"conditions as described below.

Hybridization conditions

"Low Stringency" Hybridization Conditions:

prehybridization: 5× SSC (citrate buffered salt solution known in theart, see for example Sambrook et al.), 5× Denhardt's, 0.1% SDS, 0.05%sodium pyrophosphate, 100 μg/ml sonicated salmon sperm DNA; overnight at42° C.

hybridization: prehybridization buffer was replaced by the same bufferbut including the radioactively labeled probe.

washes: 4 consecutive washes (around 30 min. each) with 2× SSC, 0.1% SDSat 37° C.:

"Intermediate Stringency" Hybridization Conditions:

prehybridization: 20% formamide, 5× SSC, 5× Denhardt's, 5 mM EDTA, 25 mMsodium phosphate (pH 6.5), 0.05% sodium pyrophosphate, 100 μg/mlsonicated salmon sperm DNA; overnight at 42° C.

hybridization: prehybridization buffer was replaced by the same bufferbut including the radioactively labeled probe.

washes: 4 consecutive washes (around 30 min. each) with 2× SSC, 0.1% SDSat 37° C.

"High Stringency" Hybridization Conditions:

prehybridization: 6× SSC, 5× Denhardt's, 0.5% SDS, 100 μ.ml⁻¹ sonicatedsalmon sperm DNA overnight at 68° C.

hybridization: 6× SSC, 5× Denhardt's, 0.5% SDS, 5 mM EDTA, 100μg.ml.sup. 1 sonicated salmon sperm DNA including the radioactivelylabeled probe.

washes: the following consecutive washes (around 30 min. each) wereperformed: --2× SSC, 0.1% SDS at room temperature (twice); and 0.1× SSC,0.1% SDS at 68° C. (twice).

EXAMPLE 6 Sequencing

All cDNAs were subcloned in pGEM7zf type vectors (Promega Corp.), andExo III deletion mutants have been generated according to methods knownin the art. Sequencing was performed using a protocol based on theSanger procedure and involving Taq polymerase and single stranded DNA onan automated 37OA DNA Sequencer.

EXAMPLE 7 Construction of plasmid "pshIL5Rα"

Plasmid constructions were carried out as described in the followingparagraphs. Unless indicated otherwise, all methods used below arestandard methodology according to Sambrook et al. (1989), MolecularCloning. A Laboratory Manual (2nd edn). Cold Spring Harbor, N.Y., ColdSpring Harbor Laboratory Press.

The insert from phage λgt11-hIL5Rα12 (see Example 5) was excised usingEcoR1 and Not1 restriction enzymes. Both sticky ends were filled inusing E. coli DNA polymerase 1 Klenow fragment in the presence of allfour deoxynucleotide triphosphates, and non-palindromic BstX1 linkerswere added using T4 DNA ligase. The sequence of these linkers is asfollows:

5'CTTAGAGCACA 3'[SEQ ID NO: 17]

3'GAAATCTC 5'. [SEQ ID NO: 18]

In the next step, the modified insert was ligated into plasmid pCDM8,see, Seed and Aruffo, Proc. Natl. Acad. Sci. USA, 84, 3365 (1987);Aruffo and Seed, Proc. Natl. Acad. Sci. USA, 84. 8573 (1987); and Seed,Nature, 329, 840 (1987); and the construct with the appropriateorientation versus the CMV-promoter was chosen for further analysis.

EXAMPLE 8 Transformation of E. coli MC1061(p3)

Transformation of E. coli MC1061 (p3) with the plasmid pshIL5Rα ofExample 7 was achieved by the electroporation procedure. A Gene Pulserfrom Bio-Rad (Richmond, Calif., USA) was used in accordance withmanufacturer's instructions, with settings at: 25 μF, 2.5 kV and 200Ohms.

EXAMPLE 9 Isolation of Plasmid DNA

Plasmid DNA from transformed E. coli MC1061, as described in Example 8,was prepared using a standard procedure Birnboim and Doly, Nucl. AcidsRes. 7, 1513 (1979); and Sambrook et al., 1989) based upon alkali lysisof the cells, followed by a cesium-chloride ultracentrifugation step. Inthis way plasmid pshIL5Rα was separated from plasmid p3. The insertcoding for shIL5Rα was cut out of pshIL5Rα and sequenced as described inExample 6. The complete nucleic acid sequence and the deduced amino acidsequence of the shIL5Rα are shown in in SEQ ID NO: 1 and SEQ ID NO: 2,respectively, SEQ ID NO: 3 is the corresponding amino acid sequence ofthe murine IL5Rα.

EXAMPLE 10 Expression of shIL5Rα in COS-1 cells

COS-1 cells were transfected using the DEAE-Dextran protocol asdescribed in Sambrook et al., 1989. Subconfluent COS-1 cells wereharvested by trypsinization and replated at 2.3×10⁴ cells/cm², 24 hoursprior to transfection. The monolayers were washed twice with minimalessential medium (MEM)-Hepes pH 7.2 and incubated for 30 minutes withthe transfection mixture [10 μg pshIL5Rα isolated as described inExample 9/0.5 mg DEAE-dextran (M_(r) =2×10⁶ ; Pharmacia, Uppsala,Sweden)/ml. MEM-Hepes, pH 7.2]. Next the cells were supplemented with 8volumes prewarmed Dulbecco's modified Eagles medium (DMEM) containing10% foetal calf serum (FCS) and 100 μM chloroquine diphosphate, andincubated for 4 hours at 37° C. Thereafter the medium was removed byaspiration and the monolayers were washed once with DMEM and incubatedfor 3 days in DMEM+10% FCS.

EXAMPLE 11 Characterization of shIL5Rα

Supernatant of COS-1 cells transfected with plasmid pshIL5Rα prepared asdescribed in Example 10 was tested for the presence of secreted shIL5Rαin a competition binding assay as follows: COS-1 cells transfected asdescribed in Example 10 with a plasmid comprising a cDNA coding formIL5Rα (for amino acid sequence see SEQ ID NO: 1), obtained from a cloneas described in Example 5 and constructed as described in Example 7,were detached by treatment with phosphate buffered saline (PBS)containing 0.5 mM EDTA and 0.02% sodium azide for 30 minutes at 37° C.,resuspended at 1.5×10⁵ cells per 0.3 ml binding medium (DMEM+10%FCS+0.02% sodium azide) and incubated with 0.8 nM ¹²⁵ I-mIL5 at 4° C.for 1 hour in the absence or presence of 100-fold excess unlabeled mIL5.Supernatant of COS-1 cells (80% of binding medium) transfected withpshIL5Rα was tested for its capacity to inhibit the binding of ¹²⁵I-mIL5. Binding was also carried out in the presence of 80% supernatantof untransfected COS-1 cells. To separate cell membrane bound 125I-mlL5from free radioactivity COS-1 cells were sedimented through a phthalateoil cushion and individual pellets were counted in a gamma counter asdescribed, for example, see Plaetinck et al., J. Exp. Med. 172, 683-691(1990).

EXAMPLE 12 Construction of a chimeric human IL5Rα-IgG1 molecule

As a first step, a polymerase chain reaction (PCR) was performed usingplasmid pshIL5Rα as a template and using the following primers:

5'-CATAGACACGACAGACACGG [SEQ ID NO: 19], located in the 5' untranslatedregion of the hIL5Rα gene (position 104→123) and

5'-TACTGCAGATCCGCCTCITGAGAACCCCACAT [SEQ ID NO: 20, a primer whichmatches the last 17 residues of the coding region of the hIL5Rα solubleform, with the addition of 15 residues coding for a Gly-Gly-Ser-Ala"linker" region, and a Pst1 recognition site. The PCR was performedusing Vent Polymerase, under conditions as described by the manufacturer(New England BioLabs Inc., Beverly, Mass., USA).

After phenol extraction and ethanol precipitation, the PCR product wasresuspended in an appropriate buffer, and was kinased by T4 kinase andblunted by Klenow Polymerase by methods described.

To the blunt ended PCR fragment, Bst X1 recognition sites were added, byligation of 2 synthetic non-palindromic oligonucleotides with thesequence

5'-CTTTAGAGCACA [SEQ ID NO: 17] and

3'-GAAATCTC [SEQ ID NO: 18].

The resulting fragment was then ligated into Bst X1-opened pCDM8 vector.

The resulting plasmid containing the fragment in a sense orientationrelative to the CMV promotor in pCDM8 was opened by Not 1 cleavage,followed by a partial Pst1 restriction digestion. A Pst1-Eag1restriction fragment was purified from the pBRHIG1 plasmid vector(Ellison et al.), and ligated into the plasmid vector described above.

The Eag1 and Not1 restriction enzymes generate the same sticky ends, andthat fusion of both causes the loss of the Not1 recognition site, butnot of the Eag1 recognition site. Hence, to favor the desiredrecombinant construct, a Not1 counterselection was performed.

EXAMPLE 13 Construction of a chimeric human IL5Rα-IgG3 molecule

The same protocol as description in Example 12 was used with thefollowing exceptions:

The PCR 5' linker was: ##STR2## which creates two extra restrictionsites as indicated 5' to the nucleotides which match with the first 6amino acids of hIL5Rα.

As PCR 3' linker the following nucleotide was used:

5'GAGCTCACCGGATCCGCCTCTTGAGAACCCCACAT. [SEQ ID NO: 22]

In addition a partial Sac1 digest was used instead of a Pst1 digest andpATHIG3(2) (Huck et al. s.a.) was used as a source of the immunoglobulingene part.

EXAMPLE 14 Construction of Plasmids

Plasmid constructions can be carried out using standard methodology asdescribed by Maniatis, et al., "Molecular Cloning: A Laboratory Manual,"Cold Spring Harbor Laboratory, New York (1982).

The starting plasmid for all constructions is designated pHT4-Y1 and canbe prepared as previously described by Traunecker, Luke and Karjalainenin Nature, 331:84-86 (1988). This vector, carrying as selection markersthe ampicillin resistance gene and the gpt gene (Mulligan and Berg.Science, 209:1422 (1980)), encodes in addition a hybrid protein composedof the whole extracellular portion of the human CD4 receptor fused tothe mouse immunoglobulin kappa light-chain constant region.

Vector pHT4-Y1 was modified by replacing the mouse Ig heavy chainpromoter by the more efficient mouse Ig kappa promoter derived from thevector pKm 1 (Traunecker et al., Eur. J. Immunol., 16:851-854 (1986)).The kappa promoter was first subcloned as a Bgl11Sa1I fragment into thevector pUC18 digested with BamHI and Sa1I, recovered as a 2.2 kb HindIII(3' recessive ends filled with the help of the Klenow fragment of E.coli DNA polymerase, called hereafter: blunt)/EcoRI fragment andsubsequently inserted into XbaI (blunt)/EcoRI digested pHT4-Y1 togenerate pHT4-YK12 (see FIG. 1).

This plasmid was then digested with BamHI, to remove the exons 3 and 4of the CD4 gene and the exon encoding the mouse Ig kappa light chainconstant region, the 3' recessive ends were filled with Klenowpolymerase and dephosphorylated with calf intestine alkaline phosphataseto facilitate the insertion of the following gene fragments (blunt endedbefore ligation):

a 3.5 kb SstI/XbaI DNA fragment containing the constant region exons C2,C3 and C4 of the mouse Ig μ heavy chain gene [Arnehim, N., et al., Cell,22:179-185 (1980)] resulting in the preliminary plasmid pCD4-Mμ*.

a 3.0 kg StuI DNA fragment containing the Hinge and the C2 and C3constant region exons of the mouse Ig γ2a heavy chain gene [Roeder, W.,et al., PNAS, 78:474-479 (1981)] resulting in the preliminary plasmidpCD4-Mγ2a*.

a HaeII DNA fragment (made blunt with T4 DNA polymerase) containing theHinge and the C2 and C3 constant region exons of the human Igγ1 heavychain gene [Ellison, J. W., et al., Nucleic Acids Res., 10:4071-4079(1982)] resulting in the preliminary plasmid pCD4-Hγ1*.

a DNA fragment containing the constant region exons C2, C3 and C4 of thehuman Ig μ heavy chain gene [Rabbits, T. H., et al., Nucleic Acids Res.,9:4509-4524 (1981)]. This DNA fragment was constructed as follows:first, the HaeII fragment of the human Ig γ1 heavy chain gene was madeblunt ended as described and inserted into a blunt ended SaII site ofpUC19 in an orientation positioning the Hinge exon towards the BamHIsite. This intermediate construct was then digested with Pst1 to removethe 3 γ1 exons except for the splice acceptor site of the Hinge exonflanking the Pst1 site in the gene (the second Pst1 fragment of thehuman μ gene containing exons C2, C3 and C4 (Rabbits, et al., seeabove). This final gene construct was then recovered by BamHI andHindIII digestion (both sites in the pUC19 polylinker), followed byKlenow treatment and insertion into vector pHT4 YK12 as described aboveresulting in the preliminary plasmid pCD4-Hμ*.

The preliminary plasmids (pCD4-Mμ*, pCD4-Mγ2a*, pCD4-Hγ1*) werecompleted by inserting a XhoI Sa1I fragment which contains the murine μheavy chain gene enhancer into the unique Sa1I site 5' to the promotor(see FIGS. 1 and 2) and the preliminary plasmid pCD4-Hμ was completed byinserting the murine μ "core" enhancer as a SmaI-PvuII fragment into theblunt-ended Sa1I site 5' to the promotor (FIG. 2), resulting in thefinal plasmids pCD4-Mμ, pCD4-Mγ2a, pCD4-Hγ1 and pCD4-Hμ. Originally theenhancer fragment XbaI EcoRI described by Gillies, et al. (Cell33:717-728, (1983)) was inserted, via an EcoRI-linker on XbaI, into theEcoRI site of the Bluescript vector (Stratagene, La Jolla, USA) in anorientation positioning the XbaI (EcorI) site next to the EcoRV site.Into the unique SstI site of the vector an adaptor was inserted whichcontained a XhoI site. Finally, the enhancer fragment was recovered withXhoI-Sa1I for constructs pCD4-Mμ, pCD4-Mγ2a and pCD4-Hγ1 or withSmaI-PvuII for construct pCD4-Hμ.

For the construction of pCD4-Hγ3 plasmid pHT4-YK12 (see FIG. 1) wasdigested with BamHI to recover the exons 3 and 4 of the CD4 gene and theexon encoding the mouse Ig kappa light chain constant region, anddephosphorylated with calf intestine alkaline phosphatase to facilitatethe insertion of a 6.0 kb Bg1II DNA fragment containing the hinge andthe C2 and C3 constant region exons of the human Igγ3 heavy chain gene[Huch, S., et al., Nucleic Acids Res., 14:1779-1789 (1986)] resulting inthe preliminary plasmid pCD4-Hγ3*. The preliminary plasmid pCD4-Hγ3* wascompleted by inserting the murine μ "cae" enhancer as described abovefor plasmid pCD4-Hμ.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 22                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1351 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (F) TISSUE TYPE: leukemia                                                     (G) CELL TYPE: promyelocytes                                                  (H) CELL LINE: HL-60                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: human HL-60                                                      (B) CLONE: lambda gt11- hIL5Ralpha12                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCGCTGCTTCTCATCGCATGGCCACCGCATTTCTCAGGCCAGGCACATTGAGCATTGGTC60                CTGTGCCTGACGCTATGCTAGATGCTGGGGTTGCAGCCACGAGCATAGACACGACAGACA120               CGGTCCTCGCCATCTTCTGTTGAGTACTGGTCGGAACAAGAGGATCGTCTGTAGACAGGC180               TACAGATTGTTTTAGATTGAAGTTTCCTGTCATGTTCACTCATCTTTAAATCCTCATAGT240               AAAAAGGATATGATCATCGTGGCGCATGTATTACTCATCCTTTTGGGGGCCACTGAGATA300               CTGCAAGCTGACTTACTTCCTGATGAAAAGATTTCACTTCTCCCACCTGTCAATTTCACC360               ATTAAAGTTACTGGTTTGGCTCAAGTTCTTTTACAATGGAAACCAAATCCTGATCAAGAG420               CAAAGGAATGTTAATCTAGAATATCAAGTGAAAATAAACGCTCCAAAAGAAGATGACTAT480               GAAACCAGAATCACTGAAAGCAAATGTGTAACCATCCTCCACAAAGGCTTTTCAGCAAGT540               GTGCGGACCATCCTGCAGAACGACCACTCACTACTGGCCAGCAGCTGGGCTTCTGCTGAA600               CTTCATGCCCCACCAGGGTCTCCTGGAACCTCAATTGTGAATTTAACTTGCACCACAAAC660               ACTACAGAAGACAATTATTCACGTTTAAGGTCATACCAAGTTTCCCTTCACTGCACCTGG720               CTTGTTGGCACAGATGCCCCTGAGGACACGCAGTATTTTCTCTACTATAGGTATGGCTCT780               TGGACTGAAGAATGCCAAGAATACAGCAAAGACACACTGGGGAGAAATATCGCATGCTGG840               TTTCCCAGGACTTTTATCCTCAGCAAAGGGCGTGACTGGCTTTCGGTGCTTGTTAACGGC900               TCCAGCAAGCACTCTGCTATCAGGCCCTTTGATCAGCTGTTTGCCCTTCACGCCATTGAT960               CAAATAAATCCTCCACTGAATGTCACAGCAGAGATTGAAGGAACTCGTCTCTCTATCCAA1020              TGGGAGAAACCAGTGTCTGCTTTTCCAATCCATTGCTTTGATTATGAAGTAAAAATACAC1080              AATACAAGGAATGGATATTTGCAGATAGAAAAATTGATGACCAATGCATTCATCTCAATA1140              ATTGATGATCTTTCTAAGTACGATGTTCAAGTGAGAGCAGCAGTGAGCTCCATGTGCAGA1200              GAGGCAGGGCTCTGGAGTGAGTGGAGCCAACCTATTTATGTGGGGTTCTCAAGATAAAGG1260              AGATAACATCCAGCTTTCCTGCCCCACACCGTATCTGAAATAAAAACAACAGCAGGGATA1320              GCAGATTAAAAAAAAAAAAAAAAAAAAAAAA1351                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (F) TISSUE TYPE: leukemia                                                     (G) CELL TYPE: Promyelocytes                                                  (H) CELL LINE: HL-60                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: human HL-60                                                      (B) CLONE: lambda gt11- hIL5Ralpha12                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetIleIleValAlaHisValLeuLeuIleLeuLeuGlyAlaThrGlu                              151015                                                                        IleLeuGlnAlaAspLeuLeuProAspGluLysIleSerLeuLeuPro                              202530                                                                        ProValAsnPheThrIleLysValThrGlyLeuAlaGlnValLeuLeu                              354045                                                                        GlnTrpLysProAsnProAspGlnGluGlnArgAsnValAsnLeuGlu                              505560                                                                        TyrGlnValLysIleAsnAlaProLysGluAspAspTyrGluThrArg                              65707580                                                                      IleThrGluSerLysCysValThrIleLeuHisLysGlyPheSerAla                              859095                                                                        SerValArgThrIleLeuGlnAsnAspHisSerLeuLeuAlaSerSer                              100105110                                                                     TrpAlaSerAlaGluLeuHisAlaProProGlySerProGlyThrSer                              115120125                                                                     IleValAsnLeuThrCysThrThrAsnThrThrGluAspAsnTyrSer                              130135140                                                                     ArgLeuArgSerTyrGlnValSerLeuHisCysThrTrpLeuValGly                              145150155160                                                                  ThrAspAlaProGluAspThrGlnTyrPheLeuTyrTyrArgTyrGly                              165170175                                                                     SerTrpThrGluGluCysGlnGluTyrSerLysAspThrLeuGlyArg                              180185190                                                                     AsnIleAlaCysTrpPheProArgThrPheIleLeuSerLysGlyArg                              195200205                                                                     AspTrpLeuSerValLeuValAsnGlySerSerLysHisSerAlaIle                              210215220                                                                     ArgProPheAspGlnLeuPheAlaLeuHisAlaIleAspGlnIleAsn                              225230235240                                                                  ProProLeuAsnValThrAlaGluIleGluGlyThrArgLeuSerIle                              245250255                                                                     GlnTrpGluLysProValSerAlaPheProIleHisCysPheAspTyr                              260265270                                                                     GluValLysIleHisAsnThrArgAsnGlyTyrLeuGlnIleGluLys                              275280285                                                                     LeuMetThrAsnAlaPheIleSerIleIleAspAspLeuSerLysTyr                              290295300                                                                     AspValGlnValArgAlaAlaValSerSerMetCysArgGluAlaGly                              305310315320                                                                  LeuTrpSerGluTrpSerGlnProIleTyrValGlyPheSerArg                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: YES                                                       (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: mouse                                                           (G) CELL TYPE: B-cell precursor                                               (H) CELL LINE: B13                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       XaaXaaXaaMetValProValLeuLeuIleLeuValGlyAlaLeuAla                              151015                                                                        ThrLeuGlnAlaAspLeuLeuAsnHisLysLysPheLeuLeuLeuPro                              202530                                                                        ProValAsnPheThrIleLysAlaThrGlyLeuAlaGlnValLeuLeu                              354045                                                                        HisTrpAspProAsnProAspGlnGluGlnArgHisValAspLeuGlu                              505560                                                                        TyrHisValLysIleAsnAlaProGlnGluAspGluTyrAspThrArg                              65707580                                                                      LysThrGluSerLysCysValThrProLeuHisGluGlyPheAlaAla                              859095                                                                        SerValArgThrIleLeuLysSerSerHisThrThrLeuAlaSerSer                              100105110                                                                     TrpValSerAlaGluLeuLysAlaProProGlySerProGlyThrSer                              115120125                                                                     ValThrAsnLeuThrCysThrThrHisThrValValSerSerHisThr                              130135140                                                                     HisLeuArgProTyrGlnValSerLeuArgCysThrTrpLeuValGly                              145150155160                                                                  LysAspAlaProGluAspThrGlnTyrPheLeuTyrTyrArgPheGly                              165170175                                                                     ValLeuThrGluLysCysGlnGluTyrSerArgAspAlaLeuAsnArg                              180185190                                                                     AsnThrAlaCysTrpPheProArgThrPheIleAsnSerLysGlyPhe                              195200205                                                                     GluGlnLeuAlaValHisIleAsnGlySerSerLysArgAlaAlaIle                              210215220                                                                     LysProPheAspGlnLeuPheSerProLeuAlaIleAspGlnValAsn                              225230235240                                                                  ProProArgAsnValThrValGluIleGluSerAsnSerLeuTyrIle                              245250255                                                                     GlnTrpGluLysProLeuSerAlaPheProAspHisCysPheAsnTyr                              260265270                                                                     GluLeuLysIleTyrAsnThrLysAsnGlyHisIleGlnLysGluLys                              275280285                                                                     LeuIleAlaAsnLysPheIleSerLysIleAspAspValSerThrTyr                              290295300                                                                     SerIleGlnValArgAlaAlaValSerSerProCysArgMetProGly                              305310315320                                                                  ArgTrpGlyGluTrpSerGlnProIleTyrValGlyLysGluArg                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TrpGlyGluTrpSerGlnProIleTyrValGlyLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCHACGTATATHGGCTGHGACCACTCHCCCCA32                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCHACGTAAATHGGCTGHGACCACTCHCCCCA32                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCHACATAGATHGGTTGHGACCATTCHCCCCA32                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCHACGTATATHGGCTGACTCCACTCHCCCCA32                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCHACGTAAATHGGCTGACTCCACTCHCCCCA32                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCHACATAGATHGGTTGGCTCCATTCHCCCCA32                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      HisValAspLeuGluTyrHisVal                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ACATGATATTCTAAATCHACATG23                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ACGTGGTACTCCAAGTCHACGTG23                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACATGATATTCHAGATCHACATG23                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACGTGGTACTCHAGGTCHACGTG23                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AATTCGCGGCCGCTTTTTTTTTTTTTTT28                                                (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CTTTAGAGCACA12                                                                (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CTCTAAAG8                                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CATAGACACGACAGACACGG20                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TACTGCAGATCCGCCTCTTGAGAACCCCACAT32                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AAGCTTGGATCCATGATCATCGTGGCGCAT30                                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GAGCTCACCGGATCCGCCTCTTGAGAACCCCACAT35                                         __________________________________________________________________________

What is claimed is:
 1. A recombinant polypeptide coded for by a DNAsequence, which DNA sequence comprises:a) a first DNA subsequenceencoding a soluble fragment of human interleukin-5 receptor alpha chaincapable of binding human interleukin-5, and b) a second DNA subsequenceencoding a constant domain of a human immunoglobulin heavy chain orlight chain.